Formation and hydrolysis of amide bonds by lipase A from Candida antarctica; exceptional features

Various commercial lyophilized and immobilized preparations of lipase A from Candida antarctica (CAL-A) were studied for their ability to catalyze the hydrolysis of amide bonds in N-acylated alpha-amino acids, 3-butanamidobutanoic acid (beta-amino acid) and its ethyl ester. The activity toward amide bonds is highly untypical of lipases, despite the close mechanistic analogy to amidases which normally catalyze the corresponding reactions. Most CAL-A preparations cleaved amide bonds of various substrates with high enantioselectivity, although high variations in substrate selectivity and catalytic rates were detected. The possible role of contaminant protein species on the hydrolytic activity toward these bonds was studied by fractionation and analysis of the commercial lyophilized preparation of CAL-A (Cat#ICR-112, Codexis). In addition to minor impurities, two equally abundant proteins were detected, migrating on SDS-PAGE a few kDa apart around the calculated size of CAL-A. Based on peptide fragment analysis and sequence comparison both bands shared substantial sequence coverage with CAL-A. However, peptides at the C-terminal end constituting a motile domain described as an active-site flap were not identified in the smaller fragment. Separated gel filtration fractions of the two forms of CAL-A both catalyzed the amide bond hydrolysis of ethyl 3-butanamidobutanoate as well as the N-acylation of methyl pipecolinate. Hydrolytic activity towards N-acetylmethionine was, however, solely confined to the fractions containing the truncated form of CAL-A. These fractions were also found to contain a trace enzyme impurity identified in sequence analysis as a serine carboxypeptidase. The possible role of catalytic impurities versus the function of CAL-A in amide bond hydrolysis is further discussed in the paper

On the curvature in logarithmic plots of rate coefficients for chemical reactions

In terms of the reduced potential energy barrier ζ = ΔuTS/kT, the rate coefficients for chemical reactions are usually expressed as proportional to e-ζ. The coupling between vibrational modes of the medium to the reaction coordinate leads to a proportionality of the regularized gamma function of Euler Q(a,ζ) = Γ(a,ζ)/Γ(a), with a being the number of modes coupled to the reaction coordinate. In this work, the experimental rate coefficients at various temperatures for several chemical reactions were fitted to the theoretical expression in terms of Q(a,ζ) to determine the extent of its validity and generality. The new expression affords lower deviations from the experimental points in 29 cases out of 38 and it accounts for the curvature in the logarithmic plots of rate coefficients versus inverse temperature. In the absence of tunneling, conventional theories predict the curvature of these plots to be identically zero

Lipase-Catalyzed Dynamic Combinatorial Resolution and the Synthesis of Heteroaromatic Cyanohydrin Ester Enantiomers

Five structurally related thiophene-based aldehydes, acetone cyanohydrin, a base, a lipase enzyme and isopropenyl acetate have been mixed in one-pot, leading to the dynamic combinatorial resolution of a cyanohydrin library. The process is applied in optimizing synthetic conditions for the cyanohydrin acetate library obtained in terms of reactivity and enantiopurity. Anhydrous Na2CO3 and lipase PS-D (Burkholderia cepacia lipase) or Novozym 435 (Candida antarctica lipase B) preparations are shown to be the best catalyst combinations for the DCR of the substrate series in toluene. The justification of the optimized process is confirmed by preparing the thiophene -based (R)-cyanohydrin acetates with 68-99% isolated yields and 82-91 % ee using the traditional dynamic kinetic resolution of each substrate

Lipase-catalyzed asymmetric acylation in the chemoenzymatic synthesis of furan-based alcohols

Eight racemic 1-(furan-2-yl)ethanols were prepared from the corresponding carbonyl compounds for enantioselective acylation studies, and seven of them were used in preparative-scale kinetic resolutions with Candida antarctica lipase B (Novozym 435) and vinyl acetate in dried diisopropyl ether. Mechanism-based competition between the (R)-acetate (enzymatic acylation product), vinyl acetate (added acylating reagent), and acetic acid (enzymatic hydrolysis product) toward CAL-B, together with the residual water of the lipase were shown to be potential reasons for side reactions, which affected the course of the kinetic resolution of 1-[5-(2-chlorophenyl) and (4-bromophenyl)furan-2-yl]ethanols. Clear effects were not observed with the other alcoholic substrates. Alcoholysis of the enantiomerically enriched (R)-acetates with methanol and CAL-B in diisopropyl ether was shown to be a potential method for the deprotection of the (R)-acetates and the formation of (R)-alcohols. (C) 2012 Elsevier Ltd. All rights reserved